Crystal structure of a lipin/Pah phosphatidic acid phosphatase

Khayyo, Valerie I.; Hoffmann, Reece M.; Wang, Huan; Bell, Justin A.; Burke, John E.; Reue, Karen; Airola, Michael V.

doi:10.1038/s41467-020-15124-z

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Published: 11 March 2020

Crystal structure of a lipin/Pah phosphatidic acid phosphatase

Nature Communications volume 11, Article number: 1309 (2020) Cite this article

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A Publisher Correction to this article was published on 02 April 2020

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Abstract

Lipin/Pah phosphatidic acid phosphatases (PAPs) generate diacylglycerol to regulate triglyceride synthesis and cellular signaling. Inactivating mutations cause rhabdomyolysis, autoinflammatory disease, and aberrant fat storage. Disease-mutations cluster within the conserved N-Lip and C-Lip regions that are separated by 500-residues in humans. To understand how the N-Lip and C-Lip combine for PAP function, we determined crystal structures of Tetrahymena thermophila Pah2 (Tt Pah2) that directly fuses the N-Lip and C-Lip. Tt Pah2 adopts a two-domain architecture where the N-Lip combines with part of the C-Lip to form an immunoglobulin-like domain and the remaining C-Lip forms a HAD-like catalytic domain. An N-Lip C-Lip fusion of mouse lipin-2 is catalytically active, which suggests mammalian lipins function with the same domain architecture as Tt Pah2. HDX-MS identifies an N-terminal amphipathic helix essential for membrane association. Disease-mutations disrupt catalysis or destabilize the protein fold. This illustrates mechanisms for lipin/Pah PAP function, membrane association, and lipin-related pathologies.

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Introduction

Triglycerides (TAG) represent the major form of long-term energy storage in eukaryotes and are synthesized by the evolutionarily conserved glycerol-3-phosphate pathway¹. The penultimate step of TAG biosynthesis is catalyzed by magnesium-dependent phosphatidic acid phosphatases (PAPs) that hydrolyze phosphatidic acid (PA) to diacylglycerol (DAG)^2,3 (Fig. 1a). There are three lipin PAPs in humans and mice^4,5 and a single PA phosphohydrolase (Pah1) in Saccharomyces cerevisiae⁶ (Fig. 1b).

**Fig. 1: Structure of Tt Pah2 reveals the N-Lip co-folds with the C-Lip to form a split immunoglobulin-like domain.**

Lipin/Pah PAPs are metabolic enzymes that regulate energy storage⁴, energy mobilization⁷, adipogenesis⁸, phospholipid synthesis^9,10, autophagy¹¹, chylomicron secretion¹⁰, and fatty acid synthesis¹². Mutations that inactivate lipin PAP function are associated with several diseases, including rhabdomyolysis^{13,14,15,16,17,18}, statin-induced myopathy^11,13, the inflammatory disorder Majeed syndrome^5,19, lipodystrophy, insulin resistance, peripheral neuropathy, and neonatal fatty liver⁴.

The architecture of lipin/Pah PAPs varies, but all PAPs conserve two regions referred to as the N-Lip and C-Lip^2,3, originally named for their location on opposite termini of human and mouse lipins⁴ (Fig. 1b). The C-Lip is predicted to contain the catalytic domain from the haloalkanoic acid dehalogenase (HAD) superfamily²⁰ and harbors a catalytic DxDxT motif^6,21. The N-Lip is required for PAP activity^21,22,23, but its function is unclear. Functional prediction for the N-Lip has been hindered by a lack of sequence homology with domains of known function and the N-Lip being uniquely found in PAPs. Separating the N-Lip and C-Lip is an extended linker that typically contains 250–500 residues^2,3. The linker sequence is highly variable, but retains a conserved function to regulate lipin/Pah PAP localization and activity^{2,22,24,25,26,27,28}.

Despite nearly 20 years of research, the role of the N-Lip and how it combines with the C-Lip to form a productive PAP enzyme has been unclear. Here we determined the first crystal structures of a lipin/Pah PAP from Tetrahymena thermophila that revealed a two-domain architecture with a HAD-like catalytic domain and an unexpected split immunoglobulin-like domain formed by the N-Lip and C-Lip. Complementary activity assays, biophysical, and cellular experiments explain how the interplay between these domains, with an N-terminal amphipathic helix, facilitates lipin membrane association and catalysis. This work also explains how disease-associated mutations inactivate this important metabolic enzyme.

Results

Minimal lipin/Pah PAPs

To establish a minimal functional core of lipin/Pah PAPs, we used domain-enhanced BLAST searches and identified lipin/Pah PAPs in some ciliate and plant species with the essential N-Lip and C-Lip regions directly fused (Supplementary Fig. 1a). These minimal lipin/Pah PAPs lack the extended linkers in mammalian lipins and S. cerevisiae Pah1 that regulate their cellular localization and activity (Fig. 1b). Six minimal lipin/Pah PAPs were cloned from cDNA or gene synthesized (Supplementary Fig. 1a). Three were tested for complementation of the growth defects of Sc pah1Δ cells on agar plates²¹. All three complemented the ability of Sc pah1Δ cells to utilize glycerol as the sole carbon source (Supplementary Fig. 1b), but none complemented the heat stress phenotype. During the course of our study, a minimal lipin/Pah from the ciliate T. thermophila (Tt) (known as Tt Pah2) was reported to also complement the glycerol phenotype, but not the heat stress phenotype of Sc pah1Δ cells^29,30. We subsequently crystallized Tt Pah2.

Tt Pah2 shares 36 and 42% sequence identity with the N-Lip and C-Lip regions of human lipins (Supplementary Fig. 2), and is an active PAP in vitro (Fig. 1c). In HEK293 cells, Tt Pah2 localized exclusively to the endoplasmic reticulum (ER) independent of fatty acid treatment, which is known to cause translocation of mammalian lipins to the ER^31,32 (Supplementary Fig. 3). The crystallized Tt Pah2 construct (residues 1–321), which lacks 14 C-terminal residues, also localized to the ER (Supplementary Fig. 3). In contrast, mouse lipin 1 localized to the nucleus under basal conditions, and partially translocated to the ER only after fatty acid treatment (Supplementary Fig. 3). This supports the hypothesis that the localization of Tt Pah2 to the ER may be constitutive, which contrasts with mammalian lipins and may relate to the presence of the linker sequence.

Overall structure

Tt Pah2 was produced in Escherichia coli and crystal structures were refined to 3.0 Å resolution in the presence of either calcium or magnesium (Table 1). Phases were determined using single-wavelength anomalous diffraction from selenomethionine-enriched protein crystallized with calcium. The structure revealed a two-domain architecture with an immunoglobulin-like domain (Ig-like domain, residues 20–139) and a catalytic domain (residues 140–321) belonging to the haloalkanoic acid dehalogenase (HAD) superfamily (Fig. 1d). Electron density was not observed for the first 19 N-terminal residues, as well as several small loops in the catalytic domain. Notably, the N-Lip did not form its own domain, but rather co-folded with the first 45 residues of the C-Lip to form the Ig-like domain, with the remainder of the C-Lip forming the HAD-like catalytic domain (Fig. 1d).

Table 1 Data collection and refinement statistics.

Full size table

Catalytic domain

The Tt Pah2 HAD-like catalytic domain is characterized by a Rossmann-like fold with two peripheral antiparallel β-strands (β6 and β7) that create a central 7 stranded mixed β-sheet, flanked by 5 α-helices (Figs. 1d, 2a). The HAD superfamily is represented by >30,000 different members that act on different substrates. The HAD superfamily commonly has large domains (~100–200 residues) inserted between topological elements referred to as “caps”, which are classified into C1 and C2-type caps. C1 cap domains are inserted after the β1 strand, while C2 cap domains are inserted after the β3 strand. In both cases, these cap domains reside above the HAD core and help form a structural pocket above the active site to generate substrate specificity^20,33.

**Fig. 2: Structural comparison of HAD and Ig domains with Tt Pah2.**

Notably, the HAD-like catalytic domain of Tt Pah2 is “capless” with an active site completely exposed to solvent (Figs. 1d, 2a) and available to interact with the membrane during interfacial catalysis. The shallow and solvent exposed active site of Tt Pah2 implies that the specificity of lipin/Pah PAPs for PA⁵ is most likely driven by interaction with the polar glycerol backbone of PA and not through interaction with the membrane-embedded fatty acid tails.

There are short peptides in Tt Pah2 inserted in the C1 and C2 cap positions that could participate in PA recognition. The C1 peptide could not be modeled due to a lack of electron density, but immediately follows the catalytic DxDxT motif and would lie in close proximity to the active site (Fig. 2a). The C2 peptide forms a helix away from the active site, near the membrane interface, but is only visible in the magnesium structure, likely due to its involvement in a crystal contact (Fig. 2a). While unlikely, the C2 peptide may be able to adopt a different conformation to participate in PA recognition.

Immunoglobulin-like domain

Adjacent to the catalytic domain is an atypical Ig-like domain that packs against and stabilizes the catalytic domain to create an expanded flat surface at the membrane interface (Fig. 1d). Canonical Ig domains have seven β-strands, labeled A–G, which form a two-layered β-sandwich (Fig. 2b). The Ig-like domain of Tt Pah2 differs significantly from canonical Ig domains. The most prominent difference occurs at the βE strand, which splays outward away from the β-sandwich core and forms a β-hairpin with a βE′ strand (Fig. 2b). A small helical insert immediately following the βE′ strand connects back to the canonical βF and βG strands to complete the β-sandwich.

Notably, the transition between the N-Lip and C-Lip occurs directly at the two-residue turn in the β-hairpin formed by the βE and βE′ strands (Fig. 2b). In Sc Pah1 and human lipins, this two-residue turn is replaced by the extended 250–500 residue linker that separates the N-Lip and C-Lip regions (Fig. S2). This implies that in human lipins and Sc Pah1, the Ig-like domain is a “split” domain that must recombine to form a functional PAP enzyme.

Catalytic mechanism

Key active site residues to hydrolyze PA to DAG are clustered at the putative membrane interface in the typical topological positions of HAD members (Fig. 3a). In the structure determined in the presence of magnesium, a Mg²⁺ ion was modeled in the typical position for HAD phosphatases with correct geometry and distances expected for Mg²⁺ coordination. This Mg²⁺ ion is coordinated by the sidechain of the first Asp residue of the DxDxT motif (Asp146), as well as by the sidechain of Asn268 and the mainchain carbonyl oxygen of Asp148. As expected in the absence of the substrate PA, the sidechain of Asp148 (the second Asp residue of the DxDxT motif) is directed away from the Mg²⁺ ion and forms a salt bridge with Arg193. Other residues predicted to stabilize the trigonal bipyramidal transition state^20,34 are present in the expected positions, with Lys244 directed towards the Mg²⁺ ion and Ser191 adjacent (Fig. 3a).

The structure suggests that lipin/Pah PAPs utilize a similar catalytic mechanism as proposed for other HAD phosphatases^20,34 with Asp146 serving as a nucleophile to attack PA and form the trigonal bipyramidal transition state, the surrounding residues stabilizing the transition state through electrostatic interactions, and Asp148 activating a water molecule for nucleophilic attack to liberate DAG and inorganic phosphate (Fig. 3c).

In the structure determined in the presence of calcium, a Ca²⁺ ion was modeled in the active site in an altered position in comparison to the Mg²⁺ ion, with Asp272 coordinating the Ca²⁺ ion, along with the sidechains of Asp146 and Asn268 (Fig. 3b). The altered location of the Ca²⁺ ion and subsequent rearrangement of key active site residues could prevent proper binding and hydrolysis of PA, which is consistent with the magnesium dependence of lipin/Pah PAPs.

Membrane association

In vitro mammalian lipins and Sc Pah1 interact strongly with liposomes containing the anionic lipid substrate, PA^24,35,36,37. Tt Pah2 also preferentially interacted with PA-containing liposomes, in comparison with neutral or anionic PS-enriched liposomes (Fig. 4a, b).

To identify the membrane-binding regions in Tt Pah2 and the conformational changes that occur during membrane interaction, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) in the absence and presence of PA-containing liposomes. The rate of deuterium exchange for the amide hydrogens is an excellent readout of secondary structure dynamics and can be used to identify regions with altered conformations between states³⁸. This technique has been particularly useful in identifying protein-membrane interfaces, and lipid-induced conformational changes^39,40.

To avoid hydrolysis of the PA substrate, HDX-MS experiments were conducted with the D146A mutant of Tt Pah2, which was catalytically inactive (Fig. 1c) even at the μM protein concentrations required for HDX-MS. HDX-MS experiments were carried out at five timepoints of exchange (3, 30, 300, 3000, 10,000 s). Critical to the analysis of an HDX-MS experiment is the generation of a peptide map that allows for localization of deuterium incorporation. A total of 193 peptides spanning 98.8% of the primary sequence were identified and quantified. All deuterium incorporation data for all timepoints can be found in the source data (Supplementary file).

HDX-MS revealed multiple regions that were protected from amide exchange in the presence of liposomes (N-terminus 1–17, 118–131, 143–158, 204–212, and 267–316) (Fig. 4c, d). As expected, several peptides around the active site were protected, consistent with their interaction with the membrane for productive catalysis (Fig. 4c, d). One peptide in the Ig-like domain was protected (Fig. 4c). This suggests the Ig-like domain contributes at least partially to membrane binding.

The most striking and largest decrease in exchange induced by membranes was within the first nine residues of the N-Lip region, which were disordered in the crystal structure (Fig. 4c–e). This substantial protection (Fig. 4d) most likely indicates formation of new secondary structure, such as an amphipathic helix. This is consistent with previous reports of an N-terminal amphipathic helix in Sc Pah1³⁵ and also suggested by a helical wheel diagram of Tt Pah2 (Fig. 4f).

As amphipathic helices commonly associate with membranes⁴¹, we speculated this region of Tt Pah2 would be critical for membrane association and PAP activity. Accordingly, deletion of the putative amphipathic helix completely eliminated membrane association with liposomes (Fig. 4a, b) and reduced the catalytic activity of Tt Pah2 by ~80% (Fig. 4g). Taken together, this suggests an N-terminal amphipathic helix is a conserved feature of lipin/Pah PAPs that drives association to PA-enriched membranes.

Structural mapping of disease mutations

Tt Pah2 conserves the native residues that are mutated in several lipin-related pathologies (Fig. 5a, Supplementary Fig. 2). Four mutations map to the HAD-like domain. Three mutations directly disrupt catalysis: both R193H (R725H in human lipin 1^17,18) and S191L (S734L in human lipin 2¹⁹) affect stages of the catalytic cycle, and G267R (G799R in human lipin 1¹⁷) lies adjacent to Asn268, which coordinates the magnesium ion (Fig. 5b, c). Y306 plays a key structural role, packing into the hydrophobic core of the HAD-like domain (Fig. 5b). Consistently, the point mutant Y306N (Y873N in mouse lipin 1⁴²) affects both the PAP activity (Fig. 5c) and overall stability of Tt Pah2 (Fig. 5d).

L103P (L635P in human lipin 1¹⁶) is the only C-Lip mutation that maps to the Ig-like domain (Fig. 5a). L103 is located in the helical insert of the Ig-like domain and makes stabilizing hydrophobic interactions with the N-Lip portion of the Ig-like domain (Fig. 5b). Mutation to proline would both break the helix and eliminate the hydrophobic interactions made by the Leu sidechain. In Tt Pah2, this mutation reduces PAP activity by 90% (Fig. 5c) and decreases the melting temperature by 5° (Fig. 5d).

G79R is analogous to the G84R missense mutation originally described in Lpin1^fld2J mice⁴, and is the only mutation in the N-Lip. Surprisingly, G79R had no affect on the stability, PAP activity, or membrane association of Tt Pah2 (Fig. 5c–f). Structurally, this glycine completes a β-turn between the βD and βE strands (Fig. 5b), with Phi and Psi angles in the expanded allowed region for glycine residues. Potential reasons for the retention of full activity for G79R are discussed in more detail below.

Catalytic unit of mammalian lipins

To support the notion that mammalian lipin PAPs function with the same domain architecture observed in Tt Pah2, we generated analogous constructs of mouse lipin 1 and mouse lipin 2 that directly fused the N-Lip and C-Lip regions (Fig. 6a). We focused on characterizing the mouse lipin 2 construct because it was more easily purified from E. coli (Supplementary Fig. 6). The mouse lipin 2 N-Lip C-Lip fusion protein retained PAP activity in vitro and was an order of magnitude more active than Tt Pah2 (Fig. 6b). Notably, a similar construct of Sc Pah1 that fuses the N-Lip and C-Lip regions has been shown to retain PAP activity in vitro, as well as maintain cellular function if a Trp-containing motif that is C-terminal to the C-Lip is retained⁴³. Together, this suggests the functional catalytic unit of lipin/Pah PAPs is represented by the domain architecture observed in Tt Pah2 with the N-Lip and C-Lip domains co-folding to form the Ig-like and HAD-like domains. In support of this, the major structural interfaces between the N-Lip and C-Lip regions are largely conserved between Tt Pah2, mammalian lipins, and Sc Pah1 (Fig. 6c) as observed by near complete sequence conservation of the core regions of the Ig-like fold, such as the βA, βA′, βB, and βB′ strands of the N-Lip (Supplementary Fig. 2b).

**Fig. 6: The N-Lip and C-Lip regions are the functional catalytic unit of lipin/Pah PAPs.**

Discussion

Prior to this study, the molecular mechanism of the N-Lip in lipin/Pah PAP function had remained an enigma for decades. Here we establish that the N-Lip co-folds with the C-Lip to form a unique and rare cytoplasmic Ig-like domain. This establishes a clear role for the N-Lip, which resides on the opposite end from the enzymatic domain in mammalian lipins, in forming a catalytically active PAP enzyme. It further establishes that the N-Lip and C-Lip are sufficient for PAP function in vitro⁴³ and suggests the elaborated and varied architecture observed in canonical lipin/Pah PAPs has evolved to allow controlled regulation of PAP localization and activity in cells.

The structure presented here almost certainly represents the active state of lipin/Pah PAPs with the Ig-like domain packing against and stabilizing the HAD-like domain. What is not clear is whether the N-Lip and C-Lip are constitutively associated in mammalian lipins and Sc Pah1 or if their interaction is regulated. It is known that posttranslational modifications within the 500-residue linker that separates the N-Lip and C-Lip in humans and yeast can regulate both lipin/Pah PAP activity and subcellular localization^{22,24,25,26,27,28}. It is therefore tempting to speculate that modifications within the linker may prevent formation of the Ig-like domain (Fig. 6d), and therefore inactivate PAP activity or prevent membrane association. Further experimentation is required to address this hypothesis.

An N-terminal amphipathic helix in Sc Pah1 was previously proposed to regulate membrane binding³⁵. Here we provide direct experimental evidence to support this structural feature with HDX-MS revealing that membranes induce significant deuterium protection of the N-terminus, which is consistent with formation of new secondary structure. This inducible formation of a membrane-binding amphipathic helix is not common, but is notably similar to the membrane sensing mechanism of the lipid biosynthetic enzyme CTP:phosphocholine cytidylyltransferase (CCT)⁴⁴. While the hydrophobic face of the amphipathic helix of Tt Pah2 clearly interacts with the membrane, it is not clear if the hydrophilic face of this helix packs against the protein or remains exposed to solvent.

Lipin/Pah PAPs are peripheral membrane enzymes that are recruited primarily to ER membranes to regulate phospholipid and triglyceride levels¹. This work further establishes a conserved lipid binding specificity of PAPs with Tt Pah2, human lipins, and Sc Pah1 all demonstrating a preference for PA-enriched membranes^24,35. This is intriguing given that PA concentration in ER membranes is quite low⁴⁵, but may reflect a regulatory feature to recruit lipins to the ER when PA concentrations are elevated beyond a certain threshold.

To our knowledge, Tt Pah2 represents the first structurally defined lipid metabolizing member of the HAD superfamily. The capless nature of the Tt Pah2 HAD domain is reminiscent of other capless HAD enzymes whose substrates are large macromolecules²⁰. Similarly, lipin/Pah PAPs require a solvent exposed active site to associate with the membrane surface during interfacial catalysis. Thus, we predict other lipid modifying HAD members will also be capless to facilitate access to their membrane-bound substrates.

Mammalian lipins are known to oligomerize into large assemblies with the N-Lip and C-Lip mediating oligomerization through N-Lip-to-N-Lip and C-Lip-to-C-Lip interactions⁴⁶. We did not observe any direct evidence of this in Tt Pah2, where the N-Lip and C-Lip regions are directly fused. It is plausible that in human lipins, the N-Lip from one lipin molecule could interact in trans with the C-Lip of a second lipin molecule to form the Ig-like domain and induce oligomerization. However, this oligomerization has not been observed in Sc Pah1. Therefore we expect mammalian lipins may contain an additional dimerization domain that enables formation of larger oligomers.

The structure of Tt Pah2 provides a much deeper understanding for the clinically relevant mutations in lipin-related pathologies. Most intriguing is the C-Lip L635P missense mutation in human Lipin1 that results in childhood rhabdomyolysis¹⁶. Here we establish that this mutation would disrupt formation of the Ig-like domain, providing a mechanism to explain lipin dysfunction in these patients. Other mutations directly affect the catalytic machinery or destabilize the HAD-like fold. The retention of full activity of the analogous mutation in Lpin1^fld2J mice was unexpected, as HA-tagged mouse lipin 1^fld2J with the G84R mutation retains only 20% activity²². This is most likely due to local differences in structure between Tt Pah2 and mouse lipin 1 near the βE strand that allows accommodation of this mutation in Tt Pah2 without affecting function.

Methods

Tt Pah2 plasmids

Residues 1–321 of the gene encoding T. thermophila Pah2 (Tt Pah2, accession number: XP_001020467.1) were codon optimized for expression in E. coli, gene synthesized (BioBasic, Canada), and inserted into pET28 and ppSUMO plasmids, which contain an N-terminal His-tag or N-terminal His-tag followed by the SUMO gene. Point mutations and truncations of Tt Pah2 were made in the ppSUMO vector by standard PCR, the QuikChange method, or overlap extension. For mammalian expression, Tt Pah2 constructs were subcloned into pcDNA3.1 (Invitrogen) with a C-terminal V5-His-tag. The 14 additional C-terminal residues in wild-type Tt Pah2 (residues 1–335) were added back to the pcDNA construct by PCR. All constructs were verified by direct sequencing.

Minimal lipin/Pah plasmids

Three minimal lipins: S. tuberosum lipin (XP_006354896), N. tabacum lipin 1a (XP_016447116), and N. tabacum lipin 1b (XP_016447115) were PCR amplified from cDNA and cloned into pET28 and ppSUMO plasmids for E. coli expression (Agilent Technologies, Cat. No. 230280). These three minimal lipins were subcloned into the yeast expression vector YEp351 that contained the endogenous 5′ and 3′ untranslated ends of the Pah1 gene by removing the Pah1 gene from the vector pGH312. Two additional minimal lipins, I. multifiliis lipin (XP_004037123, residues 1–321) and Capsicum annuum lipin (XP_016568757, residues 1–321), were codon optimized for expression in E. coli, gene synthesized (BioBasic, Canada), and inserted into pET28 and ppSUMO plasmids.

Mouse lipin 2 N-Lip C-Lip fusion plasmid

A construct of mouse lipin 2 comprising residues 1–893 Δ96-627 was cloned using the overlap extension method and cloned into ppSUMO using BamHI and NotI restriction sites.

Yeast spot assays

Saccharomyces cerevisiae GHY57 cells⁶ (Matα ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1 ura3-1 pah1 Δ::URA3) (a gift from Gil-Soo Han and George Carman) were transformed with lipin/YEp351 plasmids by the lithium acetate method and selected on agar plates with Leu- synthetic dropout (SD) media supplemented with 2% glucose at 30 °C. For spot assays, S. cerevisiae cells were grown to saturation in SD Leu- media with 2% glucose, washed with sterile water, and diluted to an optical density at 600 nm of 1. Serial dilutions (1:10) of the cells were spotted (5 μL) onto SD Leu- plates with 2% glucose or 2% glycerol, and growth was scored after 3 days of incubation at 30 °C.

Cell culture and transfection

Human embryonic kidney 293 cells (HEK293, American Type Culture Collection #CRL-1573) were maintained in minimum essential medium as specified by the supplier. All experiments were performed using cells with 2–6 passages. To investigate the subcellular location of Tt Pah2, cells were transfected with expression constructs for Tt Pah2 1–321, Tt Pah2 1–335, or full-length mouse lipin-1 with C-terminal V5 epitopes using BioT reagent according to the manufacturer’s protocol (Bioland Scientific LLC). Thirty-six hours after transfection, cells were treated with either 400 µM oleic acid or BSA (as control) for 24 h before immunostaining.

Immunostaining and confocal immunofluorescence imaging

Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min, and treated with 10% serum for 30 min at room temperature. Primary antibodies including mouse monoclonal V5 (1:500 dilution, Invitrogen, Cat. 960-25) and Calnexin (1:1000 dilution, Abcam, Cat. ab22595) were applied and incubated at 4 °C overnight, followed by incubation with relevant secondary antibodies (1:500 dilution, Alexa 647-labeled donkey-anti-mouse, Cat. A31571; Alexa 488-labeled donkey-anti-rabbit, Cat. A21206, Invitrogen) for 1 h in a dark chamber at room temperature. Coverslips were mounted using ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (Invitrogen, Cat. P36931). Images were generated with a Leica confocal SP8-STED/FLIM/FCS laser-scanning microscope equipped with an argon-krypton laser at a magnification of ×630.

Tt Pah2 overexpression and purification

Tt Pah2 plasmids were transformed into BL21 (DE3) RIPL cells (Agilent Technologies, Cat. No. 230280) for protein overexpression. Cells were grown in Terrific Broth to an OD₆₀₀ of 1.5 and then incubated with 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 15 °C overnight before harvesting. Cell pellets were lysed in buffer A comprised of 50 mM Tris pH 8, 500 mM NaCl, 60 mM imidazole, 5% glycerol, and 2 mM beta-mercaptoethanol (βME) and lysates centrifuged at 16,000 RPM. Tt Pah2 protein was purified using a HisTrap FF column and eluted in buffer B, with an increased imidazole concentration of 300 mM. Due to saturation, this purification step was repeated three times using the column flow through to maximize yield. If using the pET28 vector, the N-terminal His-tag was not cleaved and immediately followed by size exclusion chromatography. If using the ppSUMO vector, the N-terminal His-SUMO fragment was cleaved using ULP-1 protease overnight at 4 °C. Proteins were applied to a Superdex 75 26/60 HiLoad column (GE Healthcare) equilibrated with 20 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 10 mM βME, and 1 mM dithiothreitol (DTT). Purified protein was concentrated to 7–10 mg/mL, flash-frozen, and stored at −80 °C. All mutants were expressed in ppSUMO and purified using the same method (Supplementary Fig. 4).

Se-Met Tt Pah2

Selenomethionine (Se-Met) Tt Pah2 was generated by transforming the Tt Pah2/pET28 plasmid into B834 (DE3) cells (Novagen, Cat. No. 69-041-3), which are auxotrophic for methionine. An overnight culture grown in Luria Broth was added to M9 minimal media supplemented with 19 standard amino acids, l-selenomethionine (50 mg/L), and Kao and Michayluk Vitamin Solution 100× (Sigma-Aldrich). Cells were grown to OD₆₀₀ of 0.6 and induced with 100 μM IPTG at 15 °C overnight before harvesting. Se-Met Tt Pah2 was purified as described for the native protein.

Mouse lipin-2 overexpression and purification

The mouse lipin-2 N-Lip C-Lip fusion ppSUMO plasmid was transformed into BL21 (DE3) RIPL cells (Agilent Technologies, Cat. No. 230280) for protein overexpression. Cells were grown in Terrific Broth to an OD₆₀₀ of 1.5 and then incubated with 100 μM IPTG at 15 °C overnight before harvesting. Cell pellets were lysed in buffer A comprised of 50 mM Tris pH 7.5, 500 mM NaCl, 60 mM imidazole, 5% glycerol, and 2 mM βME and lysates centrifuged at 22,000 RPM. Mouse lipin-2 protein was purified using a HisTrap FF column and eluted in buffer B, with an increased imidazole concentration of 500 mM. The N-terminal His-SUMO fragment was cleaved using ULP-1 protease overnight at 4 °C. Cleaved protein was applied to a Superdex 75 26/60 HiLoad column (GE Healthcare) equilibrated with 20 mM Tris pH 7.5, 150 mM NaCl, 5% glycerol, 10 mM βME, and 1 mM DTT. Purified protein was concentrated to 1 mg/mL, flash-frozen, and stored at −80 °C.

Crystallization and data collection

Crystals of His-tagged native Tt Pah2 (10 mg/mL) were grown by hanging drop vapor diffusion using 0.2 M Ca(NO₃)₂, 15% PEG 8000, and 0.1 M MES pH 6 at room temperature. Crystals of untagged native Tt Pah2 (7 mg/mL) were grown by hanging drop vapor diffusion using 0.2 M Mg(NO₃)₂ and 10% PEG 3350. Micro-seeds using a seed-bead (Hampton research) were used to generate single crystals. Se-Met Tt Pah2 (7 mg/mL) crystals were grown in a reservoir solution of 0.2 M Ca(NO₃)₂ and 20% PEG 8000 with the addition of 1:50 diluted micro-seeds. Solutions containing 0.2 M Ca(NO₃)₂ or 0.2 M Mg(NO₃)₂, 18% PEG 8000, 0.1 M MES pH 6, and 30% glycerol were used as cryoprotectants. Native diffraction data were collected at Brookhaven National Lab NSLS II AMX beamline 17-ID-1 for untagged lipin grown in magnesium conditions, and FMX beamline 17-ID-2 for His-tagged lipin grown in calcium conditions. Se-Met SAD data were collected at the Advanced Photon Source GM CAT 23ID-B beamline at Argonne National Lab in 15° wedges. All data were processed using xia2⁴⁷ DIALS⁴⁸ in CCP4⁴⁹. Both the calcium and magnesium data sets were highly anisotropic, which is reflected in the data collection and refinement statistics.

Structure determination and refinement

Phasing was carried out in Phenix⁵⁰ using Autosol⁵¹ with 22 of the 24 Se-Met sites identified, and an initial model generated using Autobuild⁵². After manual modeling in coot⁵³ and several refinements in Phenix Refine⁵⁴, the nearly complete model was used as a search model in Phaser⁵⁵ for molecular replacement with the 3.0 Å native calcium data set. Additional model building in Coot and refinement in Phenix produced the final model (Table 1, PDB code: 6TZY). The final calcium model contained four Tt Pah2 molecules in the asymmetric unit. Electron density for three of the four Tt Pah2 molecules was well resolved with the fourth Tt Pah2 molecule (chain D) having poorly resolved electron density for portions of the HAD-like domain. The magnesium structure was phased by molecular replacement with the calcium structure, placing two molecules in the asymmetric unit. The final model was produced by manual model building in coot and refinement in Phenix (PDB code: 6TZZ). Metal ions were modeled based on the strongest positive peak in an F_O−F_C difference map after initial refinement and geometry and distance restraints were used in refinement.

PAP assay

Mixed micelles containing 10 mol% nitrobenzoxadiazole-phosphatidic acid (NBD-PA) (Cat. No. 9000341, Avanti Polar Lipids) and Triton X-100 were generated in a buffer containing 100 mM TRIS pH 7.5, 10 mM MgCl₂, 1 mM βME, and 100 mM NaCl. Micelles were incubated with 1 μg Tt Pah2 or with 7.5 ng mouse lipin 2 for 1 h at 37 °C. All enzyme assays were conducted in technical duplicates and experimental triplicates. All reactions were linear with respect to time and protein concentration (Supplementary Fig. 5). Reactions were quenched with the addition of 300 μL of HPLC grade 1:1 CHCl₃/MeOH (Sigma-Aldrich), followed by vortexing, and centrifugation at 2000 rpm. The organic phase was removed, dried under N₂(g), and resuspended with 100 μL mobile phase B. Chromatographic separation was achieved utilizing an Agilent 1100 Series HPLC. Conditions were optimized using a Peek Scientific C-8 column (3 μm particle, 3.0 × 150 mm). Mobile phase A consisted of HPLC grade water containing 0.2% formic acid (Fisher Chemical) and 1 mM ammonium formate (Sigma-Aldrich, mass spec grade). Mobile phase B consisted of HPLC grade methanol containing 0.2% formic acid and 1 mM ammonium formate.

Liposome sedimentation assay

Liposomes were made by five freeze-thaw cycles and vortexing. The liposomes were then sonicated until a homogeneous solution was achieved, generating small unilamellar liposomes. The liposomes were extruded through a 100 nm membrane (Avestin) for 15 passes to ensure size uniformity. Liposomes were composed of 20 mol% phosphatidic acid (POPA) or 20 mol% phosphatidylserine (POPS), 40 mol% phosphatidylethanolamine (POPE), and 40 or 60 mol% phosphatidylcholine (POPC) (Avanti Polar Lipids) at a final concentration of 1 mM in a buffer containing 100 mM NaCl, 50 mM Tris pH 7.5, and 1 mM βME. Fifty microliters of liposomes was mixed with 50 μL of 1 μg protein and incubated at room temperature for 20 min. Liposomes were sedimented at 55,000 rpm (Beckman TLA 100.3, 163,348 × g) for 1 h and supernatants were removed. The soluble fraction was removed, the pellet was resuspended in an equal volume of buffer, and both samples resolved on SDS-PAGE. All liposome sedimentation assays were conducted in experimental triplicates.

HDX-MS

HDX-MS reactions were performed in a similar manner as is outlined in previous publications^39,40. In brief, HDX reactions were conducted in a final reaction volume of 20 µL with a Tt Pah2 concentration of 24 pmol. Prior to the addition of deuterated solvent, the lipid containing Tt Pah2 samples were allowed to incubate with either 4 µL of 4 mg/mL lipid vesicles (20% phosphatidic acid, 60% phosphatidylcholine, 20% phosphatidylethanolamine) or 4 µL of the corresponding lipid buffer: 20 mM HEPES pH 6.5, 100 mM KCl, 10 mM MgCl₂. After a 2-min incubation period, D₂O buffer (100 mM NaCl, 20 mM Bis-Tris pH 6.5, 10 mM MgCl₂, 93% D₂O (v/v)) was added and the reaction allowed to proceed for 3, 30, 300, 3000 or 10,000 s at 18 °C before being quenched with ice-cold acidic quench buffer, resulting in a final concentration of 0.6 M guanidine-HCl and 0.9% FA post quench. All conditions and timepoints were created and run in triplicate. Samples were flash-frozen and stored at −80 °C until injection onto an ultra-performance liquid chromatography (UPLC) system for proteolytic cleavage, peptide separation, and injection onto a QTOF for mass analysis.

Protein samples were rapidly thawed and injected onto an UPLC system kept in a cold box at 2 °C. The protein was run over two immobilized pepsin columns (Applied Biosystems; Porosyme 2-3131-00) and the peptides were collected onto a VanGuard Precolumn trap (Waters). The trap was eluted in line with an ACQUITY 1.7 μm particle, 100 × 1 mm² C18 UPLC column (Waters), using a gradient of 5–36% B (Buffer A 0.1% formic acid, Buffer B 100% acetonitrile) over 16 min. Mass spectrometry (MS) experiments were performed on an Impact QTOF (Bruker) and peptide identification was done by running tandem MS (MS/MS) experiments run in data-dependent acquisition mode. The resulting MS/MS data sets were analyzed using PEAKS7 (PEAKS) and a false discovery rate was set at 1% using a database of purified proteins and known contaminants. HDExaminer Software (Sierra Analytics) was used to automatically calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state and presence of overlapping peptides. Deuteration levels were calculated using the centroid of the experimental isotope clusters. Differences in exchange for a peptide were considered significant if they met all three of the following criteria: >4% change in exchange, >0.3 Da difference in exchange, and a p value < 0.01 using a two-tailed Student’s t test. All compared samples were set within the same experiment. The full deuterium incorporation data can be found in the source data, with all data analysis parameters as mandated by the IC-HDX-MS guidelines⁵⁶ shown in Supplementary Table 1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁷ partner repository with the data set identifier PXD017575.

Thermal shift assay

Wild-type Tt Pah2 and mutants were diluted in 100 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MgCl₂ to 1 µg with 2× SYPRO Orange (Sigma-Aldrich). SYPRO orange fluorescence was measured on an RT-PCR instrument with a temperature increase of 1 °C/min. Fluorescence was normalized to the highest measured value and then fit to a nonlinear Boltzmann sigmoidal curve. Melting temperature was determined from the associated temperature of 50% normalized fluorescence. Melting temperatures of mutants were compared to wild-type Tt Pah2 to assess their effect on stability and performed in technical duplicates and experimental triplicates.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Data supporting the findings of this manuscript are available from the corresponding author upon reasonable request. A reporting summary for this Article is available as a Supplementary Information file. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6TZY and 6TZZ. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE⁵⁷ partner repository with the data set identifier PXD017575. All other data are available from the authors on request. The source data underlying Figs. 1c, 4a–e, g, 5c–f, and 6c are provided as a Source Data file.

Change history

02 April 2020
An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank the staff at the AMX and FMX (NSLS-II) and GM-CAT (APS) beamlines for assistance during data collection, George Carman and Gil-Soo Han (Rutgers) for sharing yeast strains and plasmids, Georg Jander and Pavan Kumar (Cornell) for sharing S. tuberosum cDNA, and Vitaly Citovsky and Benoit Lacroix (Stony Brook) for sharing N. tabacum cDNA. Confocal laser-scanning microscopy was performed at the Advanced Light Microscopy/Spectroscopy Laboratory and the Leica Microsystems Center of Excellence at the California NanoSystems Institute at UCLA with funding support from NIH Shared Instrumentation Grant S10OD025017 and NSF Major Research Instrumentation grant CHE-0722519. This work was supported by the National Institutes of Health grants R35 GM128666 (M.V.A.), P01 HL090553 (K.R.), and P01 HL028481 (K.R.), the American Heart Association grants 17SDG33410860 (M.V.A.), 19PRE34450192 (V.I.K.), and 18POST34060200 (H.W.), the NSERC Discovery Grant NSERC-2014-05218 (J.E.B.), the Canadian Institutes of Health Research (J.E.B., New Investigator Award), the Michael Smith Foundation for Health Research (J.E.B., Scholar Award 17686), and the Chhabra-URECA award (J.A.B.).

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Authors and Affiliations

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA
Valerie I. Khayyo, Justin A. Bell & Michael V. Airola
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada
Reece M. Hoffmann & John E. Burke
Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Huan Wang & Karen Reue

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Valerie I. Khayyo
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Reece M. Hoffmann
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Huan Wang
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Contributions

V.I.K. performed all protein purifications, crystallization experiments, liposome sedimentation assays, and enzyme assays. R.M.H. performed all HDX-MS experiments. R.M.H. and J.E.B. analyzed all HDX-MS data. H.W. performed all cellular localization studies. J.A.B. performed yeast spot assays and constructed key plasmids. V.I.K. and M.V.A. determined and refined the final crystal structures. V.I.K., K.R., J.E.B., and M.V.A. contributed intellectual and strategic input. K.R., J.E.B., and M.V.A. supervised work. V.I.K. and M.V.A. wrote the initial manuscript with contributions from R.M.H., H.W., J.E.B., and K.R. V.I.K., K.R., J.E.B., and M.V.A. edited the final manuscript. All authors approved the final manuscript.

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Correspondence to Michael V. Airola.

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Khayyo, V.I., Hoffmann, R.M., Wang, H. et al. Crystal structure of a lipin/Pah phosphatidic acid phosphatase. Nat Commun 11, 1309 (2020). https://doi.org/10.1038/s41467-020-15124-z

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Received: 03 November 2019
Accepted: 20 February 2020
Published: 11 March 2020
DOI: https://doi.org/10.1038/s41467-020-15124-z

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